A variety of assays have been developed in the art for detecting the presence, and optionally quantifying the amount, of a target nucleic acid in a sample (e.g., DNA or RNA). For example, a number of polymerase chain reaction (PCR) based assays have been developed that involve an initial step of amplifying a region of the target nucleic acid and then detecting the presence of the amplified product.
A number of hybridization assays have been developed as alternatives to PCR based assays (e.g., see Collins et al., Nucleic Acids Research, (1997) 25:2979-2984 and U.S. Pat. Nos. 5,635,352; 5,124,246; 5,681,697; 5,681,702; 5,780,610 and 5,624,802). Hybridization assays do not rely on amplification of the target nucleic acid. Instead, as illustrated in FIGS. 1 and 2, they rely on a series of complex nucleic acid hybridizations. Individual target nucleic acids are captured on a solid phase using a combination of capture probes. Some of the capture probes are bound to a solid phase (solid phase capture probes), while others are free in solution (capture extender probes). The capture extender probes have two segments. One segment hybridizes to the solid phase capture probe while the other segment hybridizes to a region of the target nucleic acid. Detection of the captured target nucleic acid is achieved using a combination of yet more probes that are free in solution. Label extender probes include segments that hybridize to a region of the target nucleic acid and the preamplifier probe portion of a nucleic acid multimer, respectively. In addition to a preamplifier probe portion, the nucleic acid multimer also includes a multiplicity of amplifier probes that are non-covalently hybridized (e.g., as shown in FIG. 1) or covalently linked (e.g., as shown in FIG. 2). The detection multimers can be branched as shown in FIGS. 1 and 2 or they can be linear. Finally, labeled oligonucleotides that hybridize to the amplifier probes of the nucleic acid multimer allow the captured target nucleic acid to be detected.
Because hybridization assays rely on such a complex and intertwined series of hybridization steps they can take a long time to complete and currently require an overnight incubation. This lengthy incubation period forces the assay to a two day process. Many clinicians and clinical laboratories that use hybridization assays (e.g., to determine HIV or HCV viral load) would significantly benefit from a one day hybridization assay. There is therefore a need in the art for methods and reagents that shorten incubation times in hybridization assays.